The aminoacyl-tRNA synthetases (aaRSs) comprise a family of up to twenty two enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the aaRSs, which guards against neurological disease. Some aaRSs have developed editing mechanisms to correct misactivated amino acids. These editing aaRSs clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyl-adenylates ("pre-transfer" of amino acid to tRNA) or misacylated aa-tRNA ("post-transfer"). Although one of these mechanisms may dominate, most aaRSs that edit appear to operate by a mixture of pre-and post-transfer editing, which has historically complicated investigations to determine their respective molecular basis. New models for leucyl-tRNA synthetase (LeuRS) have been developed to isolate pre-transfer editing activities for detailed mechanistic investigations. In addition, pre-transfer editing can be dependent on tRNA. It is hypothesized that tRNA translocation between the synthetic and editing domains controls the enzyme's pathway choice between aminoacylation, post-transfer editing, and pre-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computation, single molecule flourescence, biochemical, and molecular biology approaches to investigate tRNA translocation and the molecular basis of the enzyme- tRNA transition that forms stable aminoacylation and editing complexes. It will also characterize pre-transfer editing and its physiological impact on the cell. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs, especially the LeuRS editing site, as targets for antibiotic development. It will also enable re-engineering of aaRSs to activate alternate amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or tools in medicinal and technological applications. PUBLIC HEALTH RELEVANCE: Decreases in the fidelity of protein synthesis due to editing defects in the tRNA synthetase can result in debilitating neurological disease. In addition, the editing site of leucyl-tRNA synthetase is the target for a truly new class of antibiotics with a novel mechanism of inhibition. These antibiotics can be effectively used to address the significant rise in antibiotic resistance that has threatened human health and welfare.